Chimeric antibody with specificity to human B cell surface antigen

ABSTRACT

The present invention relates to a eukaryotic host cell that contains DNA encoding a chimeric antibody molecule comprising two heavy chains and two light chains, each of the chains comprising a variable region and a human constant region, said antibody having specificity for the antigen bound by the 2H7 murine monoclonal antibody. The invention is also related to methods of producing the antibody.

This application is a divisional of Ser. No. 665,939, filed Mar. 5,1991, now U.S. Pat. No. 5,500,362which is a continuation of Ser. No.195,961, May 13, 1988, abandoned, which is a continuation of Ser. No.016,202, filed Jan. 8, 1987, abandoned, which is a continuation-in-partof PCT/US86/02269, filed Oct. 27,1986, abandoned.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

This invention relates to recombinant DNA methods of preparing anantibody with specificity for an antigen on the surface of human Bcells, genetic sequences coding therefor, as well as methods ofobtaining such sequences.

BACKGROUND ART

The application of cell-to-cell fusion for the production of monoclonalantibodies by Kohler and Milstein (Nature (London), 256:495, 1975)spawned a revolution in biology equal in impact to that from theinvention of recombinant DNA cloning. Monoclonal antibodies producedfrom hybridomas are already widely used in clinical and basic scientificstudies. Applications of human monoclonal antibodies produced by humanhybridomas hold great promise for the treatment of cancer, vital andmicrobial infections, certain immunodeficiencies with diminishedantibody production, and other diseases and disorders of the immunesystem.

Unfortunately, a number of obstacles exist with respect to thedevelopment of human monoclonal antibodies. This is especially true whenattempting to develop therapeutically useful monoclonal antibodies whichdefine human cell surface antigens. Many of these human cell surfaceantigens are not recognized as foreign antigens by the human immunesystem; therefore, these antigens are not immunogenic in man. Bycontrast, human cellular antigens which are immunogenic in mice can beused for the production of mouse monoclonal antibodies that specificallyrecognize the human antigens. Although such antibodies may be usedtherapeutically in man, repeated injections of "foreign" antibodies,such as a mouse antibody, in humans, can lead to harmfulhypersensitivity reactions as well as increased rate of clearance of thecirculating antibody molecules so that the antibodies do not reach theirtarget site. Furthermore, mouse monoclonal antibodies may lack theability to efficiently interact with human effector cells as assessed byfunctional assays such as antibody-dependent cellular cytotoxicity(ADCC) and complement-mediated cytolysis (CDC).

Another problem faced by immunologists is that most human monoclonalantibodies obtained in cell culture are of the IgM type. When it isdesirable to obtain human monoclonals of the IgG type, however, it hasbeen necessary to use such techniques as cell sorting to identify andisolate the few cells which are producing antibodies of the IgG or othertype from the majority producing antibodies of the IgM type. A needtherefore exists for an efficient method of switching antibody classes,for any given antibody of a predetermined or desired antigenicspecificity.

The present invention bridges both the hybridoma and genetic engineeringtechnologies to provide a quick and efficient method, as well asproducts derived therefrom, for the production of a chimerichuman/non-human antibody.

The chimeric antibodies of the present invention embody a combination ofthe advantageous characteristics of monoclonal antibodies derived frommouse-mouse hybridomas and of human monoclonal antibodies. The chimericmonoclonal antibodies, like mouse monoclonal antibodies, can recognizeand bind to a human target antigen; however, unlike mouse monoclonalantibodies, the species-specific properties of the chimeric antibodieswill avoid the induction of harmful hypersensitivity reactions and mayallow for resistance to clearance when used in humans in vivo. Also, theinclusion of appropriate human immunoglobulin sequences can result in anantibody which efficiently interacts with human effector cells in vivoto cause tumor cell lysis and the like. Moreover, using the methodsdisclosed in the present invention, any desired antibody isotype can beconferred upon a particular antigen combining site.

INFORMATION DISCLOSURE STATEMENT*

Approaches to the problem of producing chimeric antibodies have beenpublished by various authors.

Morrison, S. L. et al., Proc. Natl. Acad. Sci., USA, 81:6851-6855(November 1984), describe the production of a mouse-human antibodymolecule of defined antigen binding specificity, produced by joining thevariable region genes of a mouse antibody-producing myeloma cell linewith known antigen binding specificity to human immunoglobulin constantregion genes using recombinant DNA techniques. Chimeric genes wereconstructed, wherein the heavy chain variable region exon from themyeloma cell line S107 well joined to human IgG1 or IgG2 heavy chainconstant region exons, and the light chain variable region exon from thesame myeloma to the human kappa light chain exon. These genes weretransfected into mouse myeloma cell lines and. Transformed cellsproducing chimeric mouse-human antiphosphocholine antibodies were thusdeveloped.

Morrison, S. L. et al., European Patent Publication No. 173494(published Mar. 5, 1986), disclose chimeric "receptors" (e.g.antibodies) having variable regions derived from one species andconstant regions derived from another. Mention is made of utilizing cDNAcloning to construct the genes, although no details of cDNA cloning orpriming are shown. (see pp 5, 7 and 8).

Boulianne, G. L. et al., Nature, 312:643 (Dec. 13, 1984), also producedantibodies consisting of mouse variable regions joined to human constantregions. They constructed immunoglobulin genes in which the DNA segmentsencoding mouse variable regions specific for the hapten trinitrophenyl(TNP) were joined to segments encoding human mu and kappa constantregions. These chimeric genes were expressed as functional TNP bindingchimeric IgM.

For a commentary on the work of Boulianne et al. and Morrison et al.,see Munro, Nature, 312:597 (Dec. 13, 1984), Dickson, Genetic EngineeringNews, 5, No. 3 (March 1985), or Marx, Science, 229:455 (August 1985).

Neuberger, M. S. et al., Nature, 31:268 (Mar. 25, 1985), alsoconstructed a chimeric heavy chain immunoglobulin gene in which a DNAsegment encoding a mouse variable region specific for the hapten4-hydroxy-3-nitrophenacetyl (NP) was joined to a segment encoding thehuman epsilon region. When this chimeric gene was transfected into theJ558L cell line, an antibody was produced which bound to the NP haptenand had human IgE properties.

Neuberger, M. S. et al., have also published work showing thepreparation of cell lines that secrete hapten-specific antibodies inwhich the Fc portion has been replaced either with an active enzymemoiety (Williams, G. and Neuberger, M. S. Gene 43:319, 1986) or with apolypeptide displaying c-myc antigenic determinants (Nature, 312:604,1984).

Neuberger, M. et al., PCT Publication WO 86/01533, (published Mar. 13,1986) also disclose production of chimeric antibodies (see p. 5) andsuggests, among the technique's many uses the concept of "classswitching" (see p. 6).

Taniguchi, M., in European Patent Publication No. 171 496 (publishedFeb. 19, 1986) discloses the production of chimeric antibodies havingvariable regions with tumor specificity derived from experimentalanimals, and constant regions derived from human. The correspondingheavy and light chain genes are produced in the genomic form, andexpressed in mammalian cells.

Takeda, S. et al., Nature, 314:452 (Apr. 4, 1985) have described apotential method for the construction of chimeric immunoglobulin geneswhich have intron sequences removed by the use of a retrovirus vector.However, an unexpected splice donor site caused the deletion of the Vregion leader sequence. Thus, this approach did not yield completechimeric antibody molecules.

Cabilly, S. et al., Proc. Natl. Acad. Sci., USA, 81:3273-3277 (Jun.1984), describe plasmids that direct the synthesis in E. coli of heavychains and/or light chains of anti-carcinoembryonic antigen (CEA)antibody. Another plasmid was constructed for expression of a truncatedform of heavy chain (Fd') fragment in E. coli. Functional CEA-bindingactivity was obtained by in vitro reconstitution, in E coli extracts, ofa portion of the heavy chain with light chain.

Cabilly, S., et al., European Patent Publication 125023 (published Nov.14, 1984) describes chimeric immunoglobulin genes and their presumptiveproducts as well as other modified forms. On pages 21, 28 and 33 itdiscusses cDNA cloning and priming.

Boss, M. A., European Patent Application 120694 (published Oct. 3, 1984)shows expression in E. coli of nonchimeric immunoglobulin chains with4-nitrophenyl specificity. There is a broad description of chimericantibodies but no details (see p. 9).

Wood, C. R. et al., Nature, 314:446 (April, 1985) describe plasmids thatdirect the synthesis of mouse anti-NP antibody proteins in yeast. Heavychain mu antibody proteins appeared to be glycosylated in the yeastcells. When both heavy and light chains were synthesized in the samecell, some of the protein was assembled into functional antibodymolecules, as detected by anti-NP binding activity in soluble proteinprepared from yeast cells.

Alexander, A. et al., Proc. Nat. Acad. Sci., USA, 79:3260-3264 (1982),describe the preparation of a cDNA sequence coding for an abnormallyshort human Ig gamma heavy chain (OMM gamma³ HCD serum protein)containing a 19-amino acid leader followed by the first 15 residues ofthe V region. An extensive internal deletion removes the remainder ofthe V and the entire C_(H) 1 domain. This is cDNA coding for aninternally deleted molecule.

Dolby, T. W. et al., Proc. Natl. Acad. Sci., USA, 77:6027-6031 (1980),describe the preparation of a cDNA sequence and recombinant plasmidscontaining the same coding for mu and kappa human immunoglobulinpolypeptides. One of the recombinant DNA molecules contained codons forpart of the CH₃ constant region domain and the entire 3' noncodingsequence.

Seno, M. et al., Nucleic Acids Research, 11:719-726 (1983), describe thepreparation of a cDNA sequence and recombinant plasmids containing thesame coding for part of the variable region and all of the constantregion of the human IgE heavy chain (epsilon chain).

Kurokawa, T. et al, ibid, 11:3077-3085 (1983), show the construction,using cDNA, of three expression plasmids coding for the constant portionof the human IgE heavy chain.

Liu, F. T. et al., Proc. Nat. Acad. Sci., USA, 81:5369-5373 (September1984), describe the preparation of a cDNA sequence and recombinantplasmids containing the same encoding about two-thirds of the CH₂, andall of the C_(H) 3 and C_(H) 4 domains of human IgE heavy chain.

Tsujimoto, Y. et al., Nucleic Acids Res., 12:8407-8414 (November 1984),describe the preparation of a human V lambda cDNA sequence from an Iglambda-producing human Burkitt lymphoma cell line, by taking advantageof a cloned constant region gene as a primer for cDNA synthesis.

Murphy, J., PCT Publication WO 83/03971 (published Nov. 24, 1983)discloses hybrid proteins made of fragments comprising a toxin and acell-specific ligand (which is suggested as possibly being an antibody).

Tan, et al., J. Immunol. 135:8564 (November, 1985), obtained expressionof a chimeric human-mouse immunoglobulin genomic gene after transfectioninto mouse myeloma cells.

Jones, P. T., et al., Nature 321:552 (May 1986) constructed andexpressed a genomic construct where CDR domains of variable regions froma mouse monoclonal antibody were used to substitute for thecorresponding domains in a human antibody.

Sun, L. K., et al., Hybridoma 5 suppl. 1 S17 (1986), describes achimeric human/mouse antibody with potential tumor specificity. Thechimeric heavy and light chain genes are genomic constructs andexpressed in mammalian cells.

Sahagan et al., J. Immun. 137:1066-1074 (August 1986) describe achimeric antibody with specificity to a human tumor associated antigen,the genes for which are assembled from genomic sequences.

For a recent review of the field see also Morrison, S. L., Science229:1202-1207 (Sep. 20, 1985) and Oi, V. T., et al., BioTechniques 4:214(1986).

The Oi, et al., paper is relevant as it argues that the production ofchimeric antibodies from cDNA constructs in yeast and/or bacteria is notnecessarily advantageous.

See also Commentary on page 835 in Biotechnology 4 (1986).

SUMMARY OF THE INVENTION

The invention provides a genetically engineered chimeric antibody ofdesired variable region specificity and constant region properties,through gene cloning and expression of light and heavy chains. Thecloned immunoglobulin gene products can be produced by expression ingenetically engineered cells.

The application of oligodeoxyribonucleotide synthesis, recombinant DNAcloning, and production of specific immunoglobulin chains in variousprokaryotic and eukaryotic cells provides a means for the large scaleproduction of a chimeric human/mouse monoclonal antibody withspecificity to a human B cell surface antigen.

The invention provides cDNA sequences coding for immunoglobulin chainscomprising a constant human region and a variable, non-human, region.The immunoglobulin chains can be either heavy or light.

The invention provides gene sequences coding for immunoglobulin chainscomprising a cDNA variable region of the desired specificity. These canbe combined with genomic constant regions of human origin.

The invention provides sequences as above, present in recombinant DNAmolecules in vehicles such as plasmid vectors, capable of expression indesired prokaryotic or eukaryotic hosts.

The invention provides hosts capable of producing, by culture, thechimeric antibodies and methods of using these hosts.

The invention also provides individual chimeric immunoglobulinindividual chains, as well as complete assembled molecules having humanconstant regions and variable regions with a human B cell surfaceantigen specificity, wherein both variable regions have the same bindingspecificity.

Among other immunoglobulin chains and/or molecules provided by theinvention are:

(a) a complete functional, immunoglobulin molecule comprising:

(i) two identical chimeric heavy chains comprising a variable regionwith a human B cell surface antigen specificity and human constantregion and

(ii) two identical all (i.e. non-chimeric) human light chains.

(b) a complete, functional, immunoglobulin molecule comprising:

(i) two identical chimeric heavy chains comprising a variable region asindicated, and a human constant region, and

(ii) two identical all (i.e. non-chimeric) non-human light chains.

(c) a monovalent antibody, i.e., a complete, functional immunoglobulinmolecule comprising:

(i) two identical chimeric heavy chains comprising a variable region asindicated, and a human constant region, and

(ii) two different light chains, only one of which has the samespecificity as the variable region of the heavy chains. The resultingantibody molecule binds only to one end thereof and is thereforeincapable of divalent binding.

Genetic sequences, especially cDNA sequences, coding for theaforementioned combinations of chimeric chains or of non-chimeric chainsare also provided herein.

The invention also provide for a genetic sequence, especially a cDNAsequence, coding for the variable region of desired specificity of anantibody molecule heavy and/or light chain, operably linked to asequence coding for a polypeptide different than an immunoglobulin chain(e.g., an enzyme). These sequences can be assembled by the methods ofthe invention, and expressed to yield mixed-function molecules.

The use of cDNA sequences is particularly advantageous over genomicsequences (which contain introns), in that cDNA sequences can beexpressed in bacteria or other hosts which lack appropriate RNA splicingsystems.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the DNA rearrangements and the expression of immunoglobulinmu and gamma heavy chain genes. This is a schematic representation ofthe human heavy chain gene complex (not shown to scale). Heavy chainvariable V region formation occurs through the proper joining of V_(H),D and J_(H) gene segments. This generates an active mu gene. A differentkind of DNA rearrangement called "class switching" relocates the joinedV_(H), D and J_(H) region from the vicinity of mu constant C region tothat of another heavy chain C region (switching to gamma is diagrammedhere).

FIG. 2 shows the known nucleotide sequences of human and mouse Jregions. Consensus sequences for the J regions are shown below theactual sequences. The oligonucleotide sequence below the mouse kappa Jregion consensus sequence is a Universal Immunoglobulin Gene (UIG)oligonucleotide. Note that there are only a few J regions withrelatively conserved sequences, especially near the constant regions, ineach immunoglobulin gene locus.

FIG. 3 shows the nucleotide sequences of the mouse J regions. Shownbelow are the oligonucleotide primers UIG-H (FIG. 3A) and UIG-K (FIG.3B). Note that each contains a restriction enzyme site. They can be usedas primers for the synthesis of cDNA complementary to the variableregion of mRNA, and can also be used to mutagenize, in vitro, clonedcDNA.

FIG. 4 Human Constant Domain Module. The human C gamma 1 clone, pGMH6(FIG. 4A), was isolated from the cell line GM2146. The sequence at itsJ_(H) -C_(H) 1 junction is shown. Two restriction enzyme sites areuseful as joints in recombining the C_(H) 1 gene with different V_(H)genes. The ApaI site is 16 nucleotide residues into the C_(H) 1 codingdomain of Human gamma 1; and is used in a previous construction of amouse-human chimeric heavy-chain immunoglobulin. The BstEII site is inthe J_(H) region, and is used in the construction described in thisapplication.

The human C_(K) clone, pGML60 (FIG. 4B), is a composite of two cDNAclones, one isolated from GM1500 (pK2-3), the other from GM2146 (pGML1).The J_(K) -C_(K) junction sequence shown comes from pK2-3. In vitromutagenesis using the oligonucleotide, J_(K) HindIII, was carried out toengineer a HindIII site 14 nucleotide residues 5' of the J-C junction.This changes a human GTG codon into a CTT codon.

FIG. 5 shows the nucleotide sequence of the V region of the 2H7 V_(H)cDNA clone pH2-11. The sequence was determined by the dideoxyterminationmethod using M13 subclones of gene fragments. Open circles denote aminoacid residues confirmed by peptide sequence. A sequence homologous toD_(SP).2 in the CDR3 region is underlined. The NcoI site at 5' end wasconverted to a SalI site by using SalI linkers.

FIG. 6 shows the nucleotide sequence of the V region of the 2H7 V_(K)cDNA clone pL2-12. The oligonucleotide primer used for site-directedmutagenesis is shown below the J_(K) 5 segment. Open circles denoteamino acid residues confirmed by peptide sequence.

FIG. 7 shows the construction of the light and heavy chain expressionplasmids pING2106 (FIG. 7A) and pING2101 (FIG. 7B). The SalI to BamHIfragment from pING2100 is identical to the SalI to BamHI fragment frompING2012E (see FIG. 7C). A linear representation of the circular plasmidpING2012E is shown in panel C. The 6.6 Kb SalI to BamHI fragmentcontains sequences from pSV2-neo, puc12, M8alphaRX12, and pL1. TheHindIII site in pSV2-neo was destroyed before assembly of pING2012E byHindIII cleavage, fill-in, and religation.

FIG. 8 shows the structure of several chimeric 2H7-V_(H) expressionplasmids. pING2107 is a gpt version of the light chain plasmid,pING2106. The larger ones are two-gene plasmids. pHL2-11 and pHL2-26contain both H and L genes, while pLL2-25 contains two L genes. Theywere constructed by joining an NdeI fragment containing either an H or Lgene to partially digested (with NdeI) pING2106.

FIG. 9 shows a summary of the sequence alterations made in theconstruction of the 2H7 chimeric antibody expression plasmids. Residuesunderlined in the 5' untranslated region are derived from the clonedmouse kappa and heavy-chain genes. Residues circled in the V/C boundaryresult from mutagenesis operations to engineer restriction enzyme sitesin this region.

DESCRIPTION OF THE PREFERRED EMBODIMENTS INTRODUCTION

Generally, antibodies are composed of two light and two heavy chainmolecules. Light and heavy chains are divided into domains of structuraland functional homology. The variable domains of both the light (V_(L))and the heavy (V_(H)) chains determine recognition and specificity. Theconstant region domains of light (C_(L)) and heavy (C_(H)) chains conferimportant biological properties such as antibody chain association,secretion, transplacental mobility, complement binding, and the like.

A complex series of events leads to immunoglobulin gene expression inthe antibody producing cells. The V region gene sequences conferringantigen specificity and binding are located in separate germ line genesegments called V_(H), D and J_(H) ; or V_(L) and J_(L). These genesegments are joined by DNA rearrangements to form the complete V regionsexpressed in heavy and light chains respectively (FIG. 1). Therearranged, joined (V_(L) -J_(L) and V_(H) -D-J_(H)) V segments thenencode the complete variable regions or antigen binding domains of lightand heavy chains, respectively.

DEFINITIONS

Certain terms and phrases are used throughout the specification andclaims. The following definitions are provided for purposes of clarityand consistency.

1. Expression vector--a plasmid DNA containing necessary regulatorysignals for the synthesis of mRNA derived from any gene sequence,inserted into the vector.

2. Module vector--a plasmid DNA containing a constant or variable regiongene module.

3. Expression plasmid--an expression vector that contains an insertedgene, such as a chimeric immunoglobulin gene.

4. Gene cloning--synthesis of a gene, insertion into DNA vectors,identification by hybridization, sequence analysis and the like.

5. Transfection--the transfer of DNA into mammalian cells.

GENETIC PROCESSES AND PRODUCTS

The invention provides a novel approach for the cloning and productionof a human/mouse chimeric antibody with specificity to a human B cellsurface antigen. The antigen is a polypeptide or comprises a polypeptidebound by the 2H7 monoclonal antibody described in Clark et al. Proc.Natl. Acad. Sci., U.S.A. 82:1766-1770 (1985). This antigen is aphosphoprotein designated (Bp35(CD20)) and is only expressed on cells ofthe B cell lineage. Murine monoclonal antibodies to this antigen havebeen made before and are described in Clark et al., supra; see alsoStashenko, P., et al., J. Immunol. 125:1678-1685 (1980).

The method of production combines five elements:

(1) Isolation of messenger RNA (mRNA) from the mouse hybridoma lineproducing the monoclonal antibody, cloning and cDNA productiontherefrom;

(2) Preparation of Universal Immunoglobulin Gene (UIG) oligonucleotides,useful as primers and/or probes for cloning of the variable region genesegments in the light and heavy chain mRNA from the hybridoma cell line,and cDNA production therefrom; p1 (3) Preparation of constant regiongene segment modules by cDNA preparation and cloning, or genomic genepreparation and cloning;

(4) Construction of complete heavy or light chain coding sequences bylinkage of the cloned specific immunoglobulin variable region genesegments of part (2) above to cloned human constant region gene segmentmodules;

(5) Expression and production of light and heavy

chains in selected hosts, including prokaryotic and eukaryotic cells,either in separate fermentations followed by assembly of antibodymolecules in vitro, or through production of both chains in the samecell.

One common feature of all immunoglobulin light and heavy chain genes andthe encoded messenger RNAs is the so-called J region (i.e. joiningregion, see FIG. 1). Heavy and light chain J regions have differentsequences, but a high degree of sequence homology exists (greater than80%) especially near the constant region, within the heavy J_(H) regionsor the kappa light chain J regions. This homology is exploited in thisinvention and consensus sequences of light and heavy chain J regionswere used to design oligonucleotides (designated herein as UIGs) for useas primers or probes for cloning immunoglobulin light or heavy chainmRNAs or genes (FIG. 3). Depending on the sequence of a particular UIG,it may be capable of hybridizing to all immunoglobulin mRNAs or aspecific one containing a particular J sequence. Another utility of aparticular UIG probe may be hybridization to light chain or heavy chainmRNAs of a specific constant region, such as UIG-MJK which detects allmouse J_(K) -containing sequences (FIG. 2).

UIG design can also include a sequence to introduce a restriction enzymesite into the cDNA copy of an immunoglobulin gene (see FIG. 3). Theinvention may, for example, utilize chemical gene synthesis to generatethe UIG probes for the cloning and modification of V regions fromimmunoglobulin mRNA. On the other hand, oligonucleotides can besynthesized to recognize individually, the less conserved 5'-region ofthe J regions as a diagnostic aid in identifying the particular J regionpresent in the immunoglobulin mRNA.

A multi-step procedure is utilized for generating complete V+C regioncDNA clones from the hybridoma cell light and heavy chain mRNAs. First,the complementary strand of oligodT-primed cDNA is synthesized, and thisdouble-stranded cDNA is cloned in appropriate cDNA cloning vectors suchas pBR322 (Gubler and Hoffman, Gene, 25:263 (1983)). Clones are screenedby hybridization with UIG oligonucleotide probes. Positive heavy andlight chain clones identified by this screening procedure are mapped andsequenced to select those containing V region and leader codingsequences. In vitro mutagenesis including, for example, the use of UIGoligonucleotides, is then used to engineer desired restriction enzymecleavage sites. We used this approach for the chimeric 2H7 light chain.

An expedient method is to use synthetic UIG oligonucleotides as primersfor the synthesis of cDNA. This method has the advantage ofsimultaneously introducing a desired restriction enzyme site, such asBstEII (FIG. 3) into a V region cDNA clone. We used this approach forthe chimeric 2H7 heavy chain.

Second, cDNA constant region module vectors are prepared from humancells. These cDNA clones are modified, when necessary, by site-directedmutagenesis to place a restriction site at the analogous position in thehuman sequence or at another desired location near a boundary of theconstant region. An alternative method utilizes genomic C region clonesas the source for C region module vectors.

Third, cloned V region segments generated as above are excised andligated to light or heavy chain C region module vectors. For example,one can clone the complete human kappa light chain C region and thecomplete human gamma₁ C region. In addition, one can modify the humangamma₁ region to introduce a termination codon and thereby obtain a genesequence which encodes the heavy chain portion of an F_(ab) molecule.

The coding sequences having operationally linked V and C regions arethen transferred into appropriate expression vehicles for expression inappropriate hosts, prokaryotic or eukaryotic. Operationally linked meansin-frame joining of coding sequences to derive a continuouslytranslatable gene sequence without alterations or interruptions of thetriplet reading frame.

One particular advantage of using cDNA genetic sequences in the presentinvention is the fact that they code continuously for immunoglobulinchains, either heavy or light. By "continuously" is meant that thesequences do not contain introns (i.e. are not genomic sequences, butrather, since derived from mRNA by reverse transcription, are sequencesof contiguous exons). This characteristic of the cDNA sequences providedby the invention allows them to be expressible in prokaryotic hosts,such as bacteria, or in lower eukaryotic hosts, such as yeast.

Another advantage of using cDNA cloning method is the ease andsimplicity of obtaining variable region gene modules.

The terms "constant" and "variable" are used functionally to denotethose regions of the immunoglobulin chain, either heavy or light chain,which code for properties and features possessed by the variable andconstant regions in natural non-chimeric antibodies. As noted, it is notnecessary for the complete coding region for variable or constantregions to be present, as long as a functionally operating region ispresent and available.

Expression vehicles include plasmids or other vectors. Preferred amongthese are vehicles carrying a functionally complete human constant heavyor light chain sequence having appropriate restriction sites engineeredso that any variable heavy or light chain sequence with appropriatecohesive ends can be easily inserted thereinto. Human constant heavy orlight chain sequence-containing vehicles are thus an importantembodiment of the invention. These vehicles can be used as intermediatesfor the expression of any desired complete heavy or light chain in anyappropriate host.

One preferred host is yeast. Yeast provides substantial advantages forthe production of immunoglobulin light and heavy chains. Yeasts carryout post-translational peptide modifications including glycosylation. Anumber of recombinant DNA strategies now exist which utilize strongpromoter sequences and high copy number plasmids which can be used forovert production of the desired proteins in yeast. Yeast recognizesleader sequences on cloned mammalian gene products and secretes peptidesbearing leader sequences (i.e. prepeptides) (Hitzman, et al., 11thInternational Conference on Yeast, Genetics and Molecular Biology,Montpelier, France, Sep. 13-17, 1982).

Yeast gene expression systems can be routinely evaluated for the levelof heavy and light chain production, protein stability, and secretion.Any of a series of yeast gene expression systems incorporating promoterand termination elements from the actively expressed genes coding forglycolytic enzymes produced in large quantities when yeasts are grown inmediums rich in glucose can be utilized. Known glycolytic genes can alsoprovide very efficient transcription control signals. For example, thepromoter and terminator signals of the iso-1-cytochrome C (CYC-1) genecan be utilized.

The following approach can be taken to develop and evaluate optimalexpression plasmids for the expression of cloned immunoglobulin cDNAs inyeast.

(1) The cloned immunoglobulin DNA linking V and C regions is attached todifferent transcription promoters and terminator DNA fragments;

(2) The chimeric genes are placed on yeast plasmids (see, for example,Broach, J. R. in Methods in Enzymology--Vol. 101:307 ed. Wu, R. et al.,1983));

(3) Additional genetic units such as a yeast

leader peptide may be included on immunoglobulin DNA constructs toobtain antibody secretion.

(4) A portion of the sequence, frequently the first 6 to 20 codons ofthe gene sequence may be modified to represent preferred yeast codonusage.

(5) The chimeric genes are placed on plasmids used for integration intoyeast chromosomes.

The following approaches can be taken to simultaneously express bothlight and heavy chain genes in yeast.

(1) The light and heavy chain genes are each attached to a yeastpromoter and a terminator sequence and placed on the same plasmid. Thisplasmid can be designed for either autonomous replication in yeast orintegration at specific sites in the yeast chromosome.

(2) The light and heavy chain genes are each attached to a yeastpromoter and terminator sequence on separate plasmids containingdifferent selectable markers. For example, the light chain gene can beplaced on a plasmid containing the trp1 gene as a selectable marker,while the heavy chain gene can be placed on a plasmid containing ura3 asa selectable marker. The plasmids can be designed for either autonomousreplication in yeast or integration at specific sites in yeastchromosomes. A yeast strain defective for both selectable markers iseither simultaneously or sequentially transformed with the plasmidcontaining the light chain gene and with the plasmid containing theheavy chain gene.

(3) The light and heavy chain genes are each attached to a yeastpromoter and terminator sequence on separate plasmids each containingdifferent selectable markers as described in (2) above. A yeast matingtype "a" strain defective in the selectable markers found on the lightand heavy chain expression plasmids (trp1 and ura3 in the above example)is transformed with the plasmid containing the light chain gene byselection for one of the two selectable markers (trp1 in the aboveexample). A yeast mating type "alpha" strain defective in the sameselectable markers as the "a" strain (i.e. trp1 and ura3 as examples) istransformed with a plasmid containing the heavy chain gene by selectionfor the alternate selectable marker (i.e. ura3 in the above example).The "a" strain containing the light chain plasmid (phenotype: Trp⁺ Ura⁻in the above example) and the strain containing the heavy chain plasmid(phenotype: Trp⁻ Ura⁺ in the above example) are mated and diploids areselected which are prototrophic for both of the above selectable markers(Trp⁺ Ura⁺ in the above example).

Among bacterial hosts which may be utilized as transformation hosts, E.coli K12 strain 294 (ATCC (31446) is particularly useful. Othermicrobial strains which may be used include E. coli X1776 (ATCC31537)The aforementioned strains, as well as E. coli W3110 (ATCC 27325)and other enterobacteria such as Salmonella typhimurium or Serratiamarcescens, and various Pseudomonas species may be used.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with a host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as specific genes which are capable of providingphenotypic selection in transformed cells. For example, E. coli isreadily transformed using pBR322, a plasmid derived from an E. colispecies (Bolivar, et al., Gene, 2:95 (1977)). pBR322 contains genes forampicillin and tetracycline resistance, and thus provides easy means foridentifying transformed cells. The pBR322 plasmid or other microbialplasmids must also contain, or be modified to contain, promoters whichcan be used by the microbial organism for expression of its ownproteins. Those promoters most commonly used in recombinant DNAconstruction include the beta-lactamase (penicillinase) and lactose(beta-galactosidase) promoter systems (Chang et al., Nature, 275:615(1978) Itakura et al., Science, 198:1056 (1977)) and tryptophan promotersystems (Goeddel et al., Nucleic Acids Research, 8:4057 (1980) EPOPublication No. 0036776). While these are the most commonly used, othermicrobial promoters have been discovered and utilized.

For example, a genetic construct for any heavy or light chimericimmunoglobulin chain can be placed under the control of the leftwardpromoter of bacteriophage lambda (P_(L)). This promoter is one of thestrongest known promoters which can be controlled. Control is exerted bythe lambda repressor, and adjacent restriction sites are known.

The expression of the immunoglobulin chain sequence can also be placedunder control of other regulatory sequences which may be "homologous" tothe organism in its untransformed state. For example, lactose dependentE. coli chromosomal DNA comprises a lactose or lac operon which mediateslactose digestion by elaborating the enzyme beta-galactosidase. The laccontrol elements may be obtained from bacteriophage lambda pLAC5, whichis infective for E. coli. The lac promoter-operator system can beinduced by IPTG.

Other promoter/operator systems or portions thereof can be employed aswell. For example, arabinose, colicine E1, galactose, alkalinephosphatase, tryptophan, xylose, tac, and the like can be used.

Other preferred hosts are mammalian cells, grown in vitro in tissueculture, or in vivo in animals. Mammalian cells providepost-translational modifications to immunoglobulin protein moleculesincluding leader peptide removal, correct folding and assembly of heavyand light chains, proper glycosylation at correct sites, and secretionof functional antibody protein.

Mammalian cells which may be useful as hosts for the production ofantibody proteins include cells of lymphoid origin, such as thehybridoma Sp2/0-Ag14 (ATCC CRL 1581) or the myeloma P3X63Ag8 (ATCC TIB9), and its derivatives. Others include cells of fibro-blast origin,such as Vero (ATCC CRL 81) or CHO-- K1 (ATCC CRL 61).

Several possible vector systems are available for the expression ofcloned heavy chain and light chain genes in mammalian cells. One classof vectors relies upon the integration of the desired gene sequencesinto the host cell genome. Cells which have stably integrated DNA can beselected by simultaneously introducing drug resistance genes such as E.coli gpt (Mulligan, R. C. and Berg, P., Proc. Natl. Acad. Sci., USA,78:2072 (1981)) or Tn5 neo (Southern, P. J. and Berg, P., J. Mol. Appl.Genet., 1:327 (1982)). The selectable marker gene can be either linkedto the DNA gene sequences to be expressed, or introduced into the samecell by co-transfection (Wigler, M. et al., Cell, 16:77 (1979)). Asecond class of vectors utilizes DNA elements which confer autonomouslyreplicating capabilities to an extrachromosomal plasmid. These vectorscan be derived from animal viruses, such as bovine papillomavirus(Sarver, N. et al., Proc. Natl. Acad. Sci., USA, 79:7147 (1982)),polyoma virus (Deans, R. J. et al., Proc. Natl. Acad. Sci., USA, 81:1292(1984)), or SV40 virus (Lusky, M. and Botchan, M., Nature, 293:79(1981)).

Since an immunoglobulin cDNA is comprised only of sequences representingthe mature mRNA encoding an antibody protein additional gene expressionelements regulating transcription of the gene and processing the RNA arerequired for the synthesis of immunoglobulin mRNA. These elements mayinclude splice signals, transcription promoters, including induciblepromoters enhancers, and termination signals. cDNA expression vectorsincorporating such elements include those described by Okayama, H. andBerg, P., Mol. Cell Biol., 3:280 (1983) Cepko, C. L. et al., Cell,37:1053 (1984) and Kaufman, R. J., Proc. Natl. Acad. Sci., USA, 82:689(1985).

An additional advantage of mammalian cells as hosts is their ability toexpress chimeric immunoglobulin genes which are derived from genomicsequences. Thus, mammalian cells may express chimeric immunoglobulingenes which are comprised of a variable region cDNA module plus aconstant region which is composed in whole or in part of genomicsequences. Several human constant region genomic clones have beendescribed (Ellison, J. W. et al., Nucl. Acids Res., 10:4071 (1982), orMax, E. et al., Cell, 29:691 (1982)). The use of such genomic sequencesmay be convenient the simultaneous introduction of immunoglobulinenhancers, splice signals, and transcription termination signals alongwith the constant region gene segment.

Different approaches can be followed to obtain complete H₂ L₂antibodies.

First, one can separately express the light and heavy chains followed byin vitro assembly of purified light and heavy chains into complete H₂ L₂IgG antibodies. The assembly pathways used for generation of completeH2L₂ IgG molecules in cells have been extensively studied (see, forexample, Scharff, M., Harvey Lectures, 69:125 (1974)). In vitro reactionparameters for the formation of IgG antibodies from reduced isolatedlight and heavy chains have been defined by Beychok, S., Cells ofImmunolobulin Synthesis, Academic Press, New York, page 69, 1979.

Second, it is possible to co-express light and heavy chains in the samecells to achieve intracellular association and linkage of heavy andlight chains into complete H₂ L₂ IgG antibodies. The co-expression canoccur by using either the same or different plasmids in the same host.

POLYPEPTIDE PRODUCTS

The invention provides "chimeric" immunoglobulin chains, either heavy orlight. A chimeric chain contains a constant region substantially similarto that present in a natural human immunoglobulin, and a variable regionhaving the desired antigenic specificity of the invention, i.e., to thespecified human B cell surface antigen.

The invention also provides immunoglobulin molecules having heavy andlight chains associated so that the overall molecule exhibits anydesired binding and recognition properties. Various types ofimmunoglobulin molecules are provided: monovalent, divalent, moleculeswith chimeric heavy chains and non-chimeric light chains, or moleculeswith the invention's variable binding domains attached to moietiescarrying desired functions.

Antibodies having chimeric heavy chains of the same or differentvariable region binding specificity and non-chimeric (i.e., all human orall non-human) light chains, can be prepared by appropriate associationof the needed polypeptide chains. These chains are individually preparedby the modular assembly methods of the invention.

USES

The antibodies of the invention having human constant region can beutilized for passive immunization, especially in humans, withoutnegative immune reactions such as serum sickness or anaphylactic shock.The antibodies can, of course, also be utilized in prior artimmunodiagnostic assays and kits in detectably labelled form (e.g.,enzymes, ¹²⁵ I, ¹⁴ C, fluorescent labels, etc.), or in immunobilizedform (on polymeric tubes, beads, etc.), in labelled form for in vivoimaging, wherein the label can be a radioactive emitter, or an NMRcontrasting agent such as a carbon-13 nucleus, or an X-ray contrastingagent, such as a heavy metal nucleus. The antibodies can also be usedfor in vitro localization of the antigen by appropriate labelling.

The antibodies can be used for therapeutic purposes, by themselves, incomplement mediated lysis, or coupled to toxins or therapeutic moieties,such as ricin, etc.

Mixed antibody-enzyme molecules can be used for immunodiagnosticmethods, such as ELISA. Mixed antibody-peptide effector conjugates canbe used for targeted delivery of the effector moiety with a high degreeof efficacy and specificity.

Specifically, the chimeric antibodies of this invention can be used forany and all uses in which the murine 2H7 monoclonal antibody can beused, with the obvious advantage that the chimeric ones are morecompatible with the human body.

Having now generally described the invention, the same will be furtherunderstood by reference to certain specific examples which are includedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXPERIMENTAL Materials and Methods

Tissue Culture Cell Lines

The human cell lines GM2146 and GM1500 were obtained from the HumanMutant Cell Repository (Camden, N.J.) and cultured in RPMI1640 plus 10%fetal bovine serum (M. A. Bioproducts). The cell line Sp2/0 was obtainedfrom the American Type Culture Collection and grown in Dulbecco'sModified Eagle Medium (DMEM) plus 4.5 g/1 glucose (M. A. Bioproducts)plus 10% fetal bovine serum (Hyclone, Sterile Systems, Logan, Utah).Media were supplemented with penicillin/streptomycin (Irvine Scientific,Irvine, Calif.).

Recombinant Plasmid and Bacteriophage DNAs

The plasmids pBR322, pL1 and pUC12 were purchased from Pharmacia P-LBiochemicals (Milwaukee, Wis.). The plasmids pSV2-neo and pSV2-gpt wereobtained from BRL (Gaithersburg, Md.), and are available from theAmerican Type Culture Collection (Rockville, Md.). pHu-gamma-1 is asubclone of the 8.3 Kb HindIII to BamHI fragment of the human IgG1chromosomal gene. An isolation method for of the human IgG1 chromosomalgene is described by Ellison, J. W. et al., Nucl. Acids Res., 10:4071(1982). M8alphaRX12 contains the 0.7 Kb XbaI to EcoRI fragmentcontaining the mouse heavy chain enhancer from the J-C intron region ofthe M603 chromosomal gene (Davis, M. et al., Nature, 283:733, 1979)inserted into M13mp10. DNA manipulations involving purification ofplasmid DNA by buoyant density centrifugation, restriction endonucleasedigestion, purification of DNA fragments by agarose gel electrophoresis,ligation and transformation of E. coli were as described by Maniatis, T.et al., Molecular Cloning: A Laboratory Manual, (1982) or otherprocedures. Restriction endonucleases and other DNA/RNA modifyingenzymes were purchased from Boehringer-Mannheim (Indianapolis, Ind.),BRL, New England Biolabs (Beverly, Mass.) and Pharmacia P-L.

Oligonucleotide Preparation

Oligonucleotides were either synthesized by the triester method of Itoet al. (Nucl. Acids Res., 10:1755 (1982)), or were purchased fromELESEN, Los Angeles, Calif. Tritylated, deblocked oligonucleotides werepurified on Sephadex-G50, followed by reverse-phase HPLC with a 0-25%gradient of acetonitrile in 10 mM triethylamine-acetic acid, pH 7.2, ona C18 Bondapak column (Waters Associates). Detritylation was in 80%acetic acid for 30 min., followed by evaporation thrice.Oligonucleotides were labeled with gamma-³² P!ATP by T4 polynucleotidekinase.

RNA Preparation and Analysis

Total cellular RNA was prepared from tissue culture cells by the methodof Auffray, C. and Rougeon, F. (Eur. J. Biochem., 107:303 (1980)) orChirgwin, J. M. et al. (Biochemistry, 18:5294 (1979)). Preparation ofpoly(A)⁺ RNA, methyl-mercury agarose gel electrophoresis, and "Northern"transfer to nitrocellulose were as described by Maniatis, T. et al.,supra. Total cellular RNA or poly(A)⁺ RNA was directly bound tonitrocellulose by first treating the RNA with formaldehyde (White, B. A.and Bancroft, F. C., J. Biol. Chem., 257:8569 (1982)). Hybridization tofilterbound RNA was with nick-translated DNA fragments using conditionsdescribed by Margulies, D. H. et al. (Nature, 295:168 (1982)) or with ³²P-labelled oligonucleotide using 4× SSC, 10× Denhardt's, 100 ug/mlsalmon sperm DNA at 37° C. overnight, followed by washing in 4× SSC at37° C.

cDNA Preparation and Cloning

Oligo-dT primed cDNA libraries were prepared from poly(A)⁺ RNA fromGM1500 and GM2146 cells by the methods of Land, H. et al. (Nucl. AcidsRes., 9:2251 (1981)) and Gubler, V. and Hoffman, B. J., Gene, 25:263(1983), respectively. The cDNA libraries were screened by hybridization(Maniatis, T., supra) with ³² P-labelled oligonucleotides using theprocedure of de Lange et al. (Cell, 34:891 (1983)), or withnick-translated DNA fragments.

Oligonucleotide Primer Extension and Cloning

Poly(A)⁺ RNA (20 ug) was mixed with 1.2 ug primer in 40 ul of 64 mM KCl.After denaturation at 90° C. for 5 min. and then chilling in ice, 3units Human Placental Ribonuclease Inhibitor (BRL) was added in 3 ul of1M Tris-HCl, pH 8.3. The oligonucleotide was annealed to the RNA at 42°C. for 15 minutes, then 12 ul of 0.05M DTT, 0.05M MgCl₂, and 1 mM eachof dATP, dTTP, dCTP, and dGTP was added. 2 ul of alpha-³² P-dATP (400Ci/mmol, New England Nuclear) was added, followed by 3 ul of AMV reversetranscriptase (19 units/ul, Life Sciences).

After incubation at 42° C. for 105 min., 2 ul 0.5M EDTA and 50 ul 10 mMTris, 1 mM EDTA, pH 7.6 were added. Unincorporated nucleotides wereremoved by Sephadex G-50 spin column chromatography, and the RNA-DNAhybrid was extracted with phenol, then with chloroform, and precipitatedwith ethanol. Second strand synthesis, homopolymer tailing with dGTP ordCTP, and insertion into homopolymer tailed vectors was as described byGubler and Hoffman, supra.

Site-Directed Mutagenesis

Single stranded M13 subclone DNA (1 ug) was combined with 20 ngoligonucleotide primer in 12.5 ul of Hin buffer (7 mM Tris-HCl, pH 7.6,7 mM MgCl₂, 50 mM NaCl ). After heating to 95° C. in a sealed tube, theprimer was annealed to the template by slowly cooling from 70° C. to 37°C. for 90 minutes. 2 ul dNTPs (1 mM each), 1 ul ³² P-ATP (10 uCi), 1 ulDTT (0.1M) and 0.4 ul Klenow DNA PolI (2 u, Boehringer Mannheim) wereadded and chains extended at 37° C. for 30 minutes. To this was added 1ul (10 ng) M13 reverse primer (New England Biolabs), and theheating/annealing and chain extension steps were repeated. The reactionwas stopped with 2 ul of 0.5M EDTA, pH 8, plus 80 ul of 10 mM Tris-HCl,pH 7.6, 1 mM EDTA. The products were phenol extracted and purified bySephadex G-50 spun column chromatography and ethanol precipitated priorto restriction enzyme digestion and ligation to the appropriate vector.

Transfection of Myeloma Tissue Culture Cells

The electroporation method of Potter, H. et al. (Proc. Natl. Acad. Sci.,USA, 81:7161 (1984)) was used. After transfection, cells were allowed torecover in complete DMEM for 48-72 hours, then were seeded at 10,000 to50,000 cells per well in 96-well culture plates in the presence ofselective medium. G418 (GIBCO) selection was at 0.8 mg/ml, andmycophenolic acid (Calbiochem) was at 6 ug/ml plus 0.25 mg/ml xanthine.

Assays for Immunoglobulin Synthesis and Secretion

Secreted immunoglobulin was measured directly from tissue culture cellsupernatants. Cytoplasmic protein extract was prepared by vortexing 10⁶cells in 160 ul of 1% NP40, 0.15M NaCl, 10 mM Tris, 1 mM EDTA, pH 7.6and leaving the lysate at 0° C., 15 minutes, followed by centrifugationat 10,000×g to remove insoluble debris.

A double antibody sandwich ELISA (Voller, A. et al., in Manual ofClinical Immunology, 2nd Ed., Eds. Rose, N. and Friedman, H., pp.359-371, 1980) using affinity purified antisera was used to detectspecific immunoglobulins. For detection of human IgG, the plate-boundantiserum is goat anti-human IgG (KPL, Gaithersburg, Md.) at 1/1000dilution, while the peroxidase-bound antiserum is goat anti-human IgG(KPL or Tago, Burlingame) at 1/4000 dilution. For detection of humanimmunoglobulin kappa, the plate-bound antiserum is goat anti-human kappa(Tago) at 1/500 dilution, while the peroxidase-bound antiserum is goatanti-human kappa (Cappel) at 1/1000 dilution.

EXAMPLE 1 A Chimeric Mouse-Human Immunoglobulin with Specificity for aHuman B-Cell Surface Antigen

(1) Antibody 2H7.

The 2H7 mouse monoclonal antibody (gamma 2b, kappa) recognizes a humanB-cell surface antigen, (Bp35 (CD20)) Clark, E. A., et al., Proc. Natl.Acad. Sci., U.S.A. 82:1766 (1985)). The (Bp35 (CD20)) moleculespresumably play a role in B-cell activation.

The antibody 2H7 does not react with stem cells which are progenitors ofB-cell epithelial, mesenchymal and fibroblastic cells of other organs.

(2) Identification of J Sequences in the Immunoglobulin mRNA of 2H7.

Frozen cells were thawed on ice for 10 minutes and then at roomtemperature. The suspension was diluted with 15 ml PBS and the cellswere centrifuged down. They were resuspended, after washes in PBS, in 16ml 3M LiCl, 6M urea and disrupted in a polytron shear. The preparationof mRNA and the selection of the poly(A+) fraction were carried outaccording to Auffray, C. and Rougeon, F., Eur. J. Biochem. 107:303,1980.

The poly (A+) RNA from 2H7 was hybridized individually with labeledJ_(H) 1, J_(H) 2, J_(H) 3 and J_(H) 4 oligonucleotides under conditionsdescribed by Nobrega et al. Anal. Biochem 131:141, 1983). The productswere then subjected to electrophoresis in a 1.7% agarose-TBE gel. Thegel was fixed in 10% TCA, blotted dry and exposed for autoradiography.The result showed that the 2H7 V_(H) contains J_(H) 1, J_(H) 2, or J_(H)4 but not J_(H) 3 sequences.

For the analysis of the V_(K) mRNA, the dot-blot method of White andBancroft J. Biol. Chem. 257:8569, (1982) was used. Poly (A+) RNA wasimmobilized on nitrocellulose filters and was hybridized to labeledprobe-oligonucleotides at 40° in 4× SSC. These experiments show that 2H7contains J_(K) 5 sequences.

(3) V Region cDNA Clones.

A library primed by oligo (dT) on 2H7 poly (A+) RNA was screened forkappa clones with a mouse C_(K) region probe. From the 2H7 library,several clones were isolated. A second screen with a 5' J_(K) 5 specificprobe identified the 2H7 (J_(K) 5) light-chain clones. Heavy chainclones of 2H7 were generated by priming the poly(A+) RNA with theUIGH(BstEII) oligonucleotide (see FIG. 3), and identified by screeningwith the UIGH(BstEII) oligonucleotide.

The heavy and light chain genes or gene fragments from the V_(H) andV_(K) cDNA clones pH2-11 and pL2-12 were inserted into M13 bacteriophagevectors for nucleotide sequence analysis. The complete nucleotidesequences of the variable region of these clones were determined (FIGS.5 and 6) by the dideoxy chain termination method. These sequencespredict V region amino acid compositions that agree well with theobserved compositions, and predict peptide sequences which have beenverified by direct amino acid sequencing of portions of the V regions.

The nucleotide sequences of the cDNA clones show that they areimmunoglobulin V region clones as they contain amino acid residuesdiagnostic of V domains (Kabat et al., Sequences of Proteins ofImmunological Interest; U.S. Dept of HHS, 1983).

The 2H7 V_(H) has the J_(H) 1 sequence. The 2H7 V_(L) is from the V_(K)-KpnI family (Nishi et al. Proc. Nat. Acd. Sci., USA 82:6399, 1985), anduses J_(K) 5. The cloned 2H7 V_(L) predicts an amino acid sequence whichwas confirmed by amino acid sequencing of peptides from the 2H7 lightchain corresponding to residues 81-100. The cloned 2H7 V_(H) predicts anamino acid sequence confirmed also by peptide sequencing, namelyresidues 1-12.

(4) In Vitro Mutagenesis to Engineer Restriction Enzyme Sites in the JRegion for Joining to a Human C-Module and to Remove Oligo (dC)Sequences 5' to the V Modules.

For the 2H7 V_(K), the J-region mutagenesis primer J_(K) HindIII, asshown in FIG. 6, was utilized. A human C_(K) module derived from a cDNAclone was also mutagenized to contain the HindIII sequence (see FIG. 4).The mutagenesis reaction was performed on M13 subclones of these genes.The frequency of mutant clones ranged from 0.5 to 1% of the plaquesobtained.

It had been previously observed that the oligo (dC) sequence upstream ofthe AUG codon in a V_(H) chimeric gene interferes with proper splicingin one particular gene construct. It was estimated that perhaps as muchas 70% of the RNA transcripts had undergone the mis-splicing, wherein acryptic 3' splice acceptor in the leader sequence was used. Thereforethe oligo (dC) sequence upstream of the initiator AUG was removed in allof the clones.

In one approach, an oligonucleotide was used which contains a SalIrestriction site to mutagenize the 2H7 V_(K) clone. The primer used forthis oligonucleotide-directed mutagenesis is a 22-mer which introduces aSalI site between the oligo (dC) and the initiator met codon (FIG. 6).

In a different approach, a convenient NcoI site was utilized to deletethe 5' untranslated region and oligo (dC) of the 2H7 V_(H) clone (seeFIG. 5).

The human C gamma 1 gene module is a cDNA derived from GM2146 cells(Human Genetic Mutant Cell Repository, Newark, N.J.). This C gamma 1gene module was previously combined with a mouse V_(H) gene module toform the chimeric expression plasmid pING2012E (FIG. 7C).

(5) Chimeric 2H7 Expression Plasmids.

A 2H7 chimeric heavy chain expression plasmid was derived from thereplacement of the V_(H) module of pING2012E with the V_(H) cDNA modulesto give the expression plasmid pING2101 (FIG. 7b). This plasmid directsthe synthesis of chimeric 2H7 heavy chain when transfected intomammalian cells.

For the 2H7 light chain chimeric gene, the SalI to HindIII fragment ofthe mouse V_(K) module was joined to the human C_(K) module by theprocedure outlined in FIG. 7a, forming pING2106. Replacement of the neosequence with the E. coli gpt gene derived from pSV2-gpt resulted inpING2107, which expresses 2H7 chimeric light chain and confersmycophenolic acid resistance when transfected into mammalian cells.

The inclusion of both heavy and light chain chimeric genes in the sameplasmid allows for the introduction into transfected cells of a 1:1 generatio of heavy and light chain genes leading to a balanced gene dosage.This may improve expression and decrease manipulations of transfectedcells for optimal chimeric antibody expression. For this purpose, theDNA fragments derived from the chimeric heavy and light chain genes ofpING2101 and pING2106 were combined into the expression plasmids pHL2-11and pHL2-26 (FIG. 8). This expression plasmid contains a selectableneo^(R) marker and separate transcription units for each chimeric gene,each including a mouse heavy chain enhancer.

The modifications and V-C joint regions of the 2H7 chimeric genes aresummarized in FIG. 9.

(6) Stable Transfection of Mouse Lymphoid Cells for the Production ofChimeric Antibody.

Electroporation was used (Potter et al. supra; Toneguzzo et al. Mol.Cell Biol. 6:703 1986) for the introduction of 2H7 chimeric expressionplasmid DNA into mouse Sp2/0 cells. The electroporation technique gave atransfection frequency of 10⁻⁴ ×10⁻⁵ for the Sp2/0 cells.

The expression plasmids, pING2101 and pING2106, were digested with NdeI;and the DNA was introduced into Sp2/0 cells by electroporation.Transformant 1D6 was obtained which secretes chimeric 2H7 antibody.Antibody isolated from this cell line was used for the functional assaysdone to characterize the chimeric antibody. We have also obtainedtransformants from experiments using the two-gene plasmids.

(7) Purification of Chimeric 2H7 Antibody Secreted in Tissue Culture.

a. 1D6 (Sp2/0.pING2101/pING2106.1D6) cells were grown in culture mediumDMEM (Gibco #320-1965), supplemented with 10% Fetal Bovine Serum(Hyclone #A-1111-D), 10 mM HEPES, 1× Glutamine-Pen-Strep (IrvineScientific #9316) to 1×10⁶ cell/ml.

b. The cells were then centrifuged at 400×g and resuspended inserum-free culture medium at 2×10⁶ cell/ml for 18-24 hr.

c. The medium was centrifuged at 4000 RPM n a JS-4.2 rotor (3000× g) for15 min.

d. 1.6 liter of supernatant was then filtered through a 0.45 micronfilter and then concentrated over a YM30 (Amicon Corp.) filter to 25 ml.

e. The conductance of the concentrated supernatant was adjusted to5.7-5.6 mS/cm CDM 80 radiometer and the pH was adjusted to 8.0.

f. The supernatant was centrifuged at 2000×g, 5 min., and then loadedonto a 40 ml DEAE column, which was preequilibrated with 10 mM sodiumphosphate, pH8.0.

g. The flow through fraction was collected and loaded onto a 1 mlprotein A-Sepharose (Sigma) column preequilibrated with 10 mM sodiumphosphate, pH8.0.

h. The column was washed first with 6 ml 10 mM sodium phosphate bufferpH 8.0, followed by 8 ml 0.1M sodium citrate pH 3.5, then by 6 ml 0.1Mcitric acid (pH 2.2). Fractions of 0.5 ml were collected in tubescontaining 50 ul 2M Tris base (Sigma).

i. The bulk of the IgG was in the pH 3.5 elution and was pooled andconcentrated over Centricon 30 (Amicon Corp.) to approximately 0.06 ml.

j. The buffer was changed to PBS (10 mM sodium phosphate pH 7.4, 0.15MNaCl ) in Centricon 30 by repeated diluting with PBS andreconcentrating.

k. The IgG solution was then adjusted to 0.10 ml and bovine serumalbumin (Fraction V, U.S. Biochemicals) was added to 1.0% as astabilizing reagent.

(9) Chimeric 2H7 Antibody, Like the Mouse 2H7 Antibody, SpecificallyBinds to Human B Cells.

First, the samples were tested with a binding assay, in which cells ofboth an 2H7 antigen-positive and an 2H7 antigen-negative cell line wereincubated with standard mouse monoclonal antibody 2H7 with chimeric 2H7antibody derived from the cell culture supernatants, followed by asecond reagent, fluoresceinisothiocyanate (FITC)-conjugated goatantibodies to human (or mouse, for the standard) immunoglobulin.

Binding Assays Cells from a human B cell line, T51, were used. Cellsfrom human colon carcinoma line C3347 were used as a negative control,since they, according to previous testing, do not express detectableamounts of the 2H7 antigen. The target cells were first incubated for 30min at 4° C. with either the chimeric 2H7 or with mouse 2H7 standard,which had been purified from mouse ascites. This was followed byincubation with a second, FITC-labelled, reagent, which for the chimericantibody was goat-anti-human immunoglobulin, obtained from TAGO(Burlingame, Calif.), and used at a dilution of 1:50. For the mousestandard, it was goat-anti-mouse immunoglobulin, also obtained from TAGOand used at a dilution of 1:50. Antibody binding to the cell surface wasdetermined using a Coulter Model EPIC-C cell sorter.

As shown in Table I, both the chimeric and the mouse standard 2H7 boundsignificantly, and to approximately the same extent, to the positive T51line. They did not bind above background to the 2H7 negative C-3347line.

Functional Assays

In previous studies, antibody 2H7 was tested for antibody-dependentcellular cytotoxicity (ADCC) measured by its ability to lyse ⁵¹Cr-labelled human B lymphona cells in the presence of human peripheralblood leukocytes as the source of effector cells. It was also tested forits ability to lyse ⁵¹ Cr labelled hum B cells in the presence of humanserum as the source of complement. These tests were carried out aspreviously described for mouse monoclonal anti-carcinoma antibody L6,which can mediate ADCC, as well as complement-mediated cytoxicity, CDC.The techniques used and the data described for the L6 antibody have beenpreviously described. Hellstrom, et al., Proc. Natl. Acad Sci., U.S.A.83:7059-7063 (1986).

Chimeric 2H7, but not mouse 2H7 antibody, can mediate both ADCC and CDCagainst human B lymphoma cells. Thus a hybridoma producing anon-functional mouse antibody can be converted to a hybridoma producinga chimeric antibody with ADCC and CDC activities. Such a chimericantibody is a prime candidate for the treatment or imaging of B-celldisorders, such as leukemias, lymphomas, and the like.

This invention therefore provides a method for making biologicallyfunctional antibodies when starting with a hybridoma which producesantibody which has the desired specificity for antigen but lacksbiological effector functions such as ADCC and CDC.

Conclusions

The results presented above demonstrate that the chimeric 2H7 antibodybinds to (Bp35(CD20)) antigen positive human B cells to approximatelythe same extent as the mouse 2H7 monoclonal antibody. This issignificant because the 2H7 antibody defines a surface phosphoproteinantigen (Bp35(CD20)), of about 35,000 daltons, which is expressed on thecells of B cell lineage. The 2H7 antibody does not bind detectably tovarious other cells such as fibroblasts, endothelial cells, orepithelial cells in the major organs or the stem cell precursors whichgive rise to B cells.

Although the prospect of attempting tumor therapy using monoclonalantibodies is attractive, with some partial tumor regressions beingreported, to date such monoclonal antibody therapy has been met withlimited success (Houghton et al., February 1985, Proc. Natl. Acad. Sci.,82:1242-1246). Murine monoclonal anti-(Bp35(CD20)) antibody has beenused for therapy of B cell malignancies (Press, et al.,) Blood: February1987, in press). The therapeutic efficacy of mouse monoclonal antibodies(which are the ones that have been tried so far) appears to be too lowfor most practical purposes. Because of the "human" properties which maymake the chimeric 2H7 monoclonal antibodies more resistant to clearanceand less immunogenic in vivo, the chimeric 2H7 monoclonal antibodieswill be advantageously used not only for therapy with unmodifiedchimeric antibodies, but also for development of variousimmunoconjugates with drugs, toxins, immunomodulators, isotopes, etc.,as well as for diagnostic purposes such as in vivo imaging of B-celltumors (for example, lymphomas and leukemias) using appropriatelylabelled chimeric 2H7 antibodies. Such immunoconjugation techniques areknown to those skilled in the art and can be used to modify the chimeric2H7 antibody molecules of the present invention. The chimeric 2H7antibody, by virtue of its having the human constant portion, willpossess biological activity in complement dependent and antibodydependent cytotoxicity which the mouse 2H7 does not.

An illustrative cell line secreting chimeric 2H7 antibody was depositedprior to the U.S. filing date at the ATCC, Rockville Md. This is atransfected hybridoma (corresponds to 1D6 cells supra) ATCC HB 9303.

The present invention is not to be limited in scope by the cell linesdeposited since the deposited embodiment is intended as a singleillustration of one aspect of the invention and all cell lines which arefunctionally equivalent are within the scope of the invention. Indeed,various modifications of the invention in addition to those shown in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

                  TABLE 1                                                         ______________________________________                                        Binding Assays of Chimeric 2H7 Antibody and Mouse 2H7 Mono-                   clonal Antibody to a B cell Line Expressing (Bp35(CD20)) and a                Cell Line Not Expressing This Antigen.                                        AntibodY          GAM    GAH                                                  ______________________________________                                                        Binding Ratio* for                                                            T51 B-Cells                                                   2H7 Mouse         37     Nb                                                   2H7 Chimeric      ND     29                                                   L6 Mouse           1     ND                                                                   Binding Ratio* for                                                            C3347 Cells                                                   2H7 Mouse         1.4    ND                                                   2H7 Chimeric      ND     1.3                                                  L6 Mouse          110    ND                                                   ______________________________________                                         *All assays were conducted using an antibody concentration of 10 ug/ml.       The binding ratio is the number of times brighter a test sample is than a     control sample treated with GAM(FITCConjugated goat antimouse) or GAH         (FITC conjugated goat antihuman) alone. A ratio of 1 means that the test      sample is just as bright as the control; a ratio of 2 means the test          sample is twice as bright as the control and so on.                           ND  not done                                                             

We claim:
 1. A eukaryotic host cell containing DNA encoding a chimericantibody molecule, said chimeric antibody molecule comprising two lightchains and two heavy chains and each of said chains comprising avariable region and a human constant region, said chimeric antibodybeing capable of being expressed in said eukaryotic host by said DNA,wherein said chimeric antibody has specificity for the antigen bound bythe antibody produced by hybridoma HB9303 as deposited with the ATCC,and wherein said chimeric antibody produced by said eukaryotic host cellhas a cytolytic activity that is antibody-dependent cellularcytotoxicity or complement-dependent cytolysis.
 2. The eukaryotic hostcell of claim 1, wherein said chimeric antibody has a cytolytic activitythat is antibody-dependent cellular cytotoxicity.
 3. The eukaryotic hostcell of claim 1, wherein said chimeric antibody has a cytolytic activitythat is complement-dependent cytolysis.
 4. The eukaryotic host cell ofany one of claims 1-3, wherein said host cell is a yeast cell.
 5. Theeukaryotic host cell of any one of claims 1-3, wherein said host cell isa mammalian cell.
 6. The eukaryotic host cell of any one of claims 1-3,wherein said host cell is hybridoma HB9303 as deposited with the ATCC.7. A process for producing a chimeric antibody comprising culturing aeukaryotic host cell containing DNA encoding said chimeric antibodymolecule, said chimeric antibody molecule comprising two light chainsand two heavy chains and each of said chains comprising a variableregion and a human constant region, said chimeric antibody being capableof being expressed in said eukaryotic host by said DNA, wherein saidchimeric antibody has specificity for the antigen bound by the antibodyproduced by hybridoma HB9303 as deposited with the ATCC, and whereinsaid chimeric antibody produced by said eukaryotic host cell has acytolytic activity that is antibody-dependent cellular cytotoxicity orcomplement-dependent cytolysis.
 8. The process of claim 7, wherein saidchimeric antibody has a cytolytic activity that is antibody-dependentcellular cytotoxicity.
 9. The process of claim 7, wherein said chimericantibody has a cytolytic activity that is complement-dependentcytolysis.
 10. The process of any one of claims 7-9, wherein said hostcell is a yeast cell.
 11. The process of any one of claims 7-9, whereinsaid host cell is a mammalian cell.
 12. The process of any one of claims7-9, wherein said host cell is hybridoma HB9303 as deposited with theATCC.